Fitness Penalties in the Evolution of Fungicide Resistance.

The evolution of resistance poses an ongoing threat to crop protection. Fungicide resistance provides a selective advantage under fungicide selection, but resistance-conferring mutations may also result in fitness penalties, resulting in an evolutionary trade-off. These penalties may result from the functional constraints of an evolving target site or from the resource allocation costs of overexpression or active transport. The extent to which such fitness penalties are present has important implications for resistance management strategies, determining whether resistance persists or declines between treatments, and for resistance risk assessments for new modes of action. Experimental results have proven variable, depending on factors such as temperature, nutrient status, osmotic or oxidative stress, and pathogen life-cycle stage. Functional genetics tools allow pathogen genetic background to be controlled, but this in turn raises the question of epistatic interactions. Combining fitness penalties under various conditions into a field-realistic scenario poses an important future challenge.

Heterologous expression of mutated eburicol 14alpha-demethylase (CYP51) proteins of Mycosphaerella graminicola to assess effects on azole fungicide sensitivity and intrinsic protein function.

The recent decrease in the sensitivity of the Western European population of the wheat pathogen Mycosphaerella graminicola to azole fungicides has been associated with the emergence and subsequent spread of mutations in the CYP51 gene, encoding the azole target sterol 14alpha-demethylase. In this study, we have expressed wild-type and mutated M. graminicola CYP51 (MgCYP51) variants in a Saccharomyces cerevisiae mutant carrying a doxycycline-regulatable tetO(7)-CYC promoter controlling native CYP51 expression. We have shown that the wild-type MgCYP51 protein complements the function of the orthologous protein in S. cerevisiae. Mutant MgCYP51 proteins containing amino acid alterations L50S, Y459D, and Y461H and the two-amino-acid deletion DeltaY459/G460, commonly identified in modern M. graminicola populations, have no effect on the capacity of the M. graminicola protein to function in S. cerevisiae. We have also shown that the azole fungicide sensitivities of transformants expressing MgCYP51 variants with these alterations are substantially reduced. Furthermore, we have demonstrated that the I381V substitution, correlated with the recent decline in the effectiveness of azoles, destroys the capacity of MgCYP51 to complement the S. cerevisiae mutant when introduced alone. However, when I381V is combined with changes between residues Y459 and Y461, the function of the M. graminicola protein is partially restored. These findings demonstrate, for the first time for a plant pathogenic fungus, the impacts that naturally occurring CYP51 alterations have on both azole sensitivity and intrinsic protein function. In addition, we also provide functional evidence underlying the order in which CYP51 alterations in the Western European M. graminicola population emerged.

Evolutionary bi-stability in pathogen transmission mode.

Many pathogens transmit to new hosts by both infection (horizontal transmission) and transfer to the infected host's offspring (vertical transmission). These two transmission modes require specific adaptations of the pathogen that can be mutually exclusive, resulting in a trade-off between horizontal and vertical transmission. We show that in mathematical models such trade-offs can lead to the simultaneous existence of two evolutionary stable states (evolutionary bi-stability) of allocation of resources to the two modes of transmission. We also show that jumping between evolutionary stable states can be induced by gradual environmental changes. Using quantitative PCR-based estimates of abundance in seed and vegetative parts, we show that the pathogen of wheat, Phaeosphaeria nodorum, has jumped between two distinct states of transmission mode twice in the past 160 years, which, based on published evidence, we interpret as adaptation to environmental change. The finding of evolutionary bi-stability has implications for human, animal and other plant diseases. An ill-judged change in a disease control programme could cause the pathogen to evolve a new, and possibly more damaging, combination of transmission modes. Similarly, environmental changes can shift the balance between transmission modes, with adverse effects on human, animal and plant health.

Long-term relationships between environment and abundance in wheat of Phaeosphaeria nodorum and Mycosphaerella graminicola.

Relationships between weather, agronomic factors and wheat disease abundance were examined to determine possible causes of variability on century time scales. In archived samples of wheat grain and leaves obtained from the Rothamsted Broadbalk experiment archive (1844-2003), amounts of wheat, Phaeosphaeria nodorum and Mycosphaerella graminicola DNA were determined by quantitative polymerase chain reaction (PCR). Relationships between amounts of pathogens and environmental and agronomic factors were examined by multiple regression. Wheat DNA decayed at approx. 1% yr(-1) in stored grain. No M. graminicola DNA was detected in grain samples. Fluctuations in amounts of P. nodorum in grain were related to changes in spring rainfall, summer temperature and national SO(2) emission. Differences in amounts of P. nodorum between grain and leaf were related to summer temperature and spring rainfall. In leaves, annual variation in spring rainfall affected both pathogens similarly, but SO(2) had opposite effects. Previous summer temperature had a highly significant effect on M. graminicola. Cultivar effects were significant only at P = 0.1. Long-term variation in P. nodorum and M. graminicola DNA in leaf and grain over the period 1844-2003 was dominated by factors related to national SO(2) emissions. Annual variability was dominated by weather factors occurring over a period longer than the growing season.

A novel substitution I381V in the sterol 14alpha-demethylase (CYP51) of Mycosphaerella graminicola is differentially selected by azole fungicides.

SUMMARY The recent reduction in the efficacy of azole fungicides in controlling Septoria leaf blotch of wheat, caused by Mycosphaerella graminicola, has prompted concerns over possible development of resistance, particularly in light of the recent emergence of widespread resistance to quinone outside inhibitors (QoIs). We have recently implicated alterations in the target-encoding sterol 14alpha-demethylase protein (CYP51), and over-expression of genes encoding efflux pumps, in reducing sensitivity to the azole class of sterol demethylation inhibitors (DMIs) in M. graminicola. Here we report on the prevalence and selection of two CYP51 alterations, substitution I381V and deletion of codons 459 and 460 (DeltaY459/G460), in populations of M. graminicola. Neither alteration has previously been identified in human or plant pathogenic fungi resistant to azoles. The presence of DeltaY459/G460 showed a continuous distribution of EC(50) values across isolates with either I381 or V381, and had no measurable effect on azole sensitivity. Data linking fungicide sensitivity with the presence of I381V in M. graminicola show for the first time that a particular CYP51 alteration is differentially selected by different azoles in field populations of a plant pathogen. Substitution I381V although not an absolute requirement for reduced azole sensitivity, is selected by tebuconazole and difenoconazole treatment, suggesting an adaptive advantage in the presence of these two compounds. Prochloraz treatments appeared to select negatively for I381V, whereas other azole treatments did not or only weakly impacted on the prevalence of this substitution. These findings suggest treatments with different members of the azole class of fungicides could offer a resistance management strategy.

Application of real-time and multiplex polymerase chain reaction assays to study leaf blotch epidemics in barley.

ABSTRACT Leaf blotch, caused by Rhynchosporium secalis, was studied in a range of winter barley cultivars using a combination of traditional plant pathological techniques and newly developed multiplex and real-time polymerase chain reaction (PCR) assays. Using PCR, symptomless leaf blotch colonization was shown to occur throughout the growing season in the resistant winter barley cv. Leonie. The dynamics of colonization throughout the growing season were similar in both Leonie and Vertige, a susceptible cultivar. However, pathogen DNA levels were approximately 10-fold higher in the susceptible cultivar, which expressed symptoms throughout the growing season. Visual assessments and PCR also were used to determine levels of R. secalis colonization and infection in samples from a field experiment used to test a range of winter barley cultivars with different levels of leaf blotch resistance. The correlation between the PCR and visual assessment data was better at higher infection levels (R(2) = 0.81 for leaf samples with >0.3% disease). Although resistance ratings did not correlate well with levels of disease for all cultivars tested, low levels of infection were observed in the cultivar with the highest resistance rating and high levels of infection in the cultivar with the lowest resistance rating.

Impact of changes in the target P450 CYP51 enzyme associated with altered triazole-sensitivity in fungal pathogens of cereal crops.

Control of diseases caused by fungi in both medicine and agriculture is heavily dependent on the use of triazoles. As a consequence, resistance to triazoles is a threat to both human health and the sustainability of agricultural production systems. In human pathogens, particularly Candida albicans, mutations encoding alterations in the target cytochrome P450 sterol 14alpha-demethylase (CYP51; where CYP is cytochrome P450) enzyme are the primary determinants of triazole resistance. In fungal pathogens of cereals, CYP51A1 modifications, some at positions known to contribute to a resistant phenotype in human pathogens, have also been identified in isolates with altered triazole-sensitivity. However, unlike medicine where resistance to triazoles is a major clinical problem, failures of triazoles to control crop diseases in the field are rare with mean population sensitivities generally remaining low, perhaps due to differences in the selection pressures imposed on human and cereal pathogen populations. Nonetheless, the biological potential for resistance exists, and the question remains as to whether widespread triazole resistance can develop in an important cereal pathogen.

Role of Ascospores in Further Spread of QoI-Resistant Cytochrome b Alleles (G143A) in Field Populations of Mycosphaerella graminicola.

ABSTRACT Strobilurin fungicides or quinone outside inhibitors (QoIs) have been used successfully to control Septoria leaf blotch in the United Kingdom since 1997. However, QoI-resistant isolates of Mycosphaerella graminicola were reported for the first time at Rothamsted during the summer of 2002. Sequence analysis of the cytochrome b gene revealed that all resistant isolates carried a mutation resulting in the replacement of glycine by alanine at codon 143 (G143A). Extensive monitoring using real-time polymerase chain reaction (PCR) testing revealed that fungicide treatments based on QoIs rapidly selected for isolates carrying resistant A143 (R) alleles within field populations. This selection is driven mainly by polycyclic dispersal of abundantly produced asexual conidia over short distances. In order to investigate the role of sexually produced airborne ascospores in the further spread of R alleles, a method integrating spore trapping with real-time PCR assays was developed. This method enabled us to both quantify the number of M. graminicola ascospores in air samples as well as estimate the frequency of R alleles in ascospore populations. As expected, most ascospores were produced at the end of the growing season during senescence of the wheat crop. However, a rapid increase in R-allele frequency, from 35 to 80%, was measured immediately in airborne ascospore populations sampled in a wheat plot after the first QoI application at growth stage 32. After the second QoI application, most R-allele frequencies measured for M. graminicola populations present in leaves and aerosols sampled from the treated plot exceeded 90%. Spatial sampling and testing of M. graminicola flag leaf populations derived from ascospores in the surrounding crop showed that ascospores carrying R alleles can spread readily within the crop at distances of up to 85 m. After harvest, fewer ascospores were detected in air samples and the R-allele frequencies measured were influenced by ascospores originating from nearby wheat fields.

Exploring infection of wheat and carbohydrate metabolism in Mycosphaerella graminicola transformants with differentially regulated green fluorescent protein expression.

A Mycosphaerella graminicola strain transformed with the green fluorescent protein (GFP) downstream of either a carbon source-repressed promoter or a constitutive promoter was used to investigate in situ carbohydrate uptake during penetration of the fungus in wheat leaves. The promoter region of the acu-3 gene from Neurospora crassa encoding isocitrate lyase was used as a carbon source-repressed promoter. The promoter region of the Aspergillus nidulans gpdA gene encoding glyceraldehyde-3-phosphate dehydrogenase was used as a constitutive promoter. Fluorometric measurement of GFP gene expression in liquid cultures of acu-3-regulated transformants indicated that the N. crassa acu-3 promoter functions in M. graminicola as it does in N. crassa, i.e., acetate induced and carbon source repressed. Glucose, fructose, and saccharose triggered the repression, whereas mannitol, xylose, and cell wall polysaccharides did not. Monitoring the GFP level during fungal infection of wheat leaves revealed that acu-3 promoter repression occurred after penetration until sporulation, when newly differentiated pycnidiospores fluoresced. The use of GFP transformants also allowed clear visualization of M. graminicola pathogenesis. No appressoria were formed, but penetration at cell junctions was observed. These results give new insight into the biotrophic status of M. graminicola.

Occurrence and molecular characterization of strobilurin resistance in cucumber powdery mildew and downy mildew.

ABSTRACT Between 1998 and 1999, control failure of powdery mildew (Podosphaera fusca) and downy mildew (Pseudoperonospora cubensis) by the strobilurin fungicides azoxystrobin and kresoxim-methyl was observed in cucumber-growing areas of Japan. Results from inoculation tests carried out on intact cucumber plants and leaf disks clearly showed the distribution of pathogen isolates highly resistant to azoxystrobin and kresoximmethyl. Fragments of the fungicide-targeted mitochondrial cytochrome b gene were polymerase chain reaction amplified from total pathogen DNA and their sequences analyzed to elucidate the molecular mechanism of resistance. A single point mutation (GGT to GCT) in the cytochrome b gene, resulting in substitution of glycine by alanine at position 143, was found in resistant isolates of downy mildew. This substitution in cytochrome b seemed to result in high resistance to strobilurins in this pathogen. The same mutation was found in some but not all resistant isolates of powdery mildew. This study suggests that a mutation at position 143 in the target-encoding gene, resulting in an amino acid substitution, was probably a major cause of the rapid development of high strobilurin resistance in these two pathogens.

Analysis of conductance responses during depolymerization of pectate by soft rot Erwinia spp. and other pectolytic bacteria isolated from potato tubers.

Different bacteria isolated from potato tubers were screened for their pectolytic properties by examining pitting in polypectate agar, recording conductance responses in polypectate medium and performing potato tuber soft rot tests. For bacteria found positive in conductimetry, the role of polygalacturonase (PG) and pectate lyase (PL) in the generation of conductance changes in a polygalacturonic acid (PGA) medium was further analysed using enzyme activity staining after gel electrophoresis and high-performance anion exchange chromatography. The extent of the conductance changes during depolymerization of PGA was dependent on the amounts of galacturonate monomers and oligomers accumulated in the medium. In comparison with an unidentified saprophyte and a Klebsiella strain, both mainly having PL activity, soft rot Erwinia spp. rapidly produced larger conductance responses, due to a combined action of multiple forms of PG and PL. The responses of Erwinia spp. were initially associated with the accumulation of large amounts of monomers and saturated dimers to heptamers, due to PG activity. Subsequently, as well as monomers and saturated dimers, large amounts of unsaturated dimers were also detected, due to PL activity. The role of PG as an important conductimetric factor was also demonstrated for a pectinase preparation derived from Aspergillus niger. Besides detection, automated conductimetric assays in pectate media may also be useful for monitoring of pectolytic activity in pectinase preparations and for screening of pectolytic activity of microorganisms under different media and growth conditions.